Expression of Recombinant Subunit Protein of Pasteurella multocida toxin in Escherichia coli

• Main Authors: Yi-wen Lin, 林怡妏
• Other Authors: Ming-Jiuan Wu
• Format: Others
• Language: zh-TW
• Published: 2008
• Online Access: http://ndltd.ncl.edu.tw/handle/26247362870224298168

碩士 === 嘉南藥理科技大學 === 生物科技研究所 === 96 === In this study, the effects of culture conditions on the growth of recombinant E. coli BL21 (DE3) and the expression of subunit protein of Pasteurella multocida toxin 1 (Tox1) were investigated in both shaking flask and fermentor. In shaking flask, the optimal condition for the expression of Tox1 was by culturing E. coli in LB medium at 37oC with initial pH 6.17-7.87 until mid-log phase, which is about 3-4 h after inoculation. Inducer, IPTG (2.5~400 �嵱), was then administrated and cells were cultured at 25-37oC for another 3- 7 h and the yield of Tox1 could be as high as 0.1 g/L. Three different media, i.e. SSP, modified R and high density culture (HCDC), were used to study the optimal condition in fermentor in dissolved oxygen-stat (DO-stat) culture. It was found that cell density of 15 OD600 units and 0.5 g/L Tox1 (50-60 mg/L/h) could be obtained when recombinant E. coli was cultured in SSP medium at 37oC until mid-log phase followed by induction and culture with IPTG (100-200�n�嵱) at 25oC. Higher cell density of 53 OD600 units and 1.5 g/L Tox1 (169 mg/L/h) could further be achieved by changing to modified R culture with similar culture/induction parameters. The highest yield which was cell density of 180 OD600 units and 5.8 g/L Tox1 (370 mg/L/h), was obtained when recombinant E. coli was cultured in HCDC with feeding. In contradict to previous literature, the majority of recombinant Tox1 (74%) was expressed in soluble form inside E. coli. Only 28% of the soluble Tox1 could be purified by precipitation with 10-30% ammonium sulfate. The purified Tox1, however, was degraded into 74 kDa and 66 kDa fragments so that no exact PI could be determined.